Microbiology 47 (2015) 12e20

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Food Microbiology

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Effect of lipolytic activity of Candida adriatica, Candida diddensiae and Yamadazyma terventina on the acidity of extra-virgin with a different polyphenol and content

* G. Ciafardini, B.A. Zullo

Department of Agricultural, Environmental and Food Sciences, University of Molise, Via De Sanctis, I-86100, Campobasso, Italy article info abstract

Article history: Previous microbiological research demonstrated the presence of a rich micro-flora composed mainly of Received 31 July 2014 yeasts in the suspended fraction of freshly produced olive oil. Some of the yeasts are considered harmful Received in revised form as they can damage the quality of the olive oil through the hydrolysis of the triacylglycerols. Present 22 October 2014 research has demonstrated that the lipolytic activity of some lipase-producer strains belonging to a yeast Accepted 31 October 2014 species called Candida adriatica, Candida diddensiae and Yamadazyma terventina can be modulated by the Available online 11 November 2014 water and the polyphenol content of olive oil. Laboratory tests highlighted a substantial increase in free in the inoculated olive oil characterized by high water content and low polyphenol concen- Keywords: tration. The acidity of the olive oil samples containing 0.06% and 0.31% of water increased significantly by Extra-virgin olive oil Olive oil acidity 33% in the lipase-producer yeast strains tested during a period of 2 weeks of incubation at 30 C. All other Polyphenols yeasts showed strong lipolytic activity in the presence of 1.31% of water e the only exception to this was Yeast the C. adriatica 1985 strain. The phenolic compounds typical of olive oil represent another important Water content in olive oil factor able to condition the viability and the lipolytic activity of the lipase-producer yeasts. From the tests performed on the olive oil characterized by an increasing content of total polyphenols equal to 84, 150 and 510 mg per kg of oil, the percentage of the lipase-producer yeasts able to hydrolyse the tri- acylglycerols was respectively 100%, 67% and 11%. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction aqueous and non-aqueous (i.e. olive oil) phases and this feature distinguishes them from esterases (Macre and Hammond, 1985). Olive oil represents the main source used in the The lipases hydrolyse the ester-carboxylate bonds of acylglycerols Mediterranean area and its use is now spreading rapidly to other producing free fatty acids, partial acylglycerols and glycerol. The areas such as the U.S. and Australia. Extra virgin olive oil is obtained lipases originating from the fruits are the main cause of the acidity from the fruits of the olive tree using an exclusively mechanical of newly produced olive oil coming from olives damaged before process and is one of the oldest edible oils that can be consumed processing (Frega et al., 1999). However, recent microbiological without any refining process. The quality of olive oil based on research has proved that freshly produced olive oil is contaminated chemical parameters includes the presence of free fatty acids. In by capable of conditioning the physical-chemical line with European Community regulation 299/2013 (EC, 2013), less and organoleptic characteristics of the oil through the production than 0.8% of total free fatty acids, expressed as oleic acid, is required of enzymes (Ciafardini and Zullo, 2002a, b; Zullo et al., 2013). The in the production of extra-virgin olive oil. A hydrolase family of microorganisms found in olive oil are mainly those from yeasts enzymes, the lipase, are involved in the production of the free fatty originating from the olives' phyllosphera. During the processing of acids in olive oil. The lipase (EC-3.1.1.3) catalyses the hydrolysis of the olives in the mill they migrate into the oil where they reproduce water-insoluble ester such as triacylglycerols. These enzymes in a selective way depending on the chemical composition of the oil possess the unique feature of acting as an interface between the and go on to constitute the typical microbiota of each oil (Ciafardini et al., 2004). Some of these microorganisms were found to be beneficial as they produce a b-glucosidase and esterase capable of

* Corresponding author. Tel.: þ39 874 404689; fax: þ39 874 404855. increasing the quality of the more bitter olive oil through the hy- E-mail address: [email protected] (B.A. Zullo). drolysis of the glucoside known as oleuropein into simpler and no http://dx.doi.org/10.1016/j.fm.2014.10.010 0740-0020/© 2014 Elsevier Ltd. All rights reserved. G. Ciafardini, B.A. Zullo / Food Microbiology 47 (2015) 12e20 13 longer bitter compounds such as hydroxytyrosol and elenolic acid 30 C. At the end of incubation a series of masters were set up with characterized also by a high antioxidant activity. Nevertheless, the yeast strains using the CHROMagar Candida medium (BBL other forms of yeasts that increase acidity through the production 4354093, Madison, USA). Transfer of the colonies on CHROMagar of lipase during olive oil storage can compromise oil quality. Lipase Candida medium was carried out using small wooden sticks steri- activity has been noticed in many oil-born yeast forms of Candida lised in an autoclave at 120 C for 30 min. Both the colony wickerhamii and Candida parapsilosis species (Zullo and Ciafardini, morphology and their colour were evaluated after 7 days incuba- 2008b). Other research, carried out on the distribution of dimor- tion at 30 C as described by Tornai-Lehoczki et al. (2003). The phic yeast species in commercial extra-virgin olive oil, showed both influence of nutritional stress condition on the growth behaviour of þ the presence of lipase-producer (Lip ) and Lip strains of Candida the above-reported dimorphic yeasts was evaluated by considering diddensiae in different olive oil samples (Zullo et al., 2010, 2013). the appearance of the pseudohyphae during the growth phase in a However, olive oil is a heterogeneous habitat where yeasts with poor medium represented by the corn-meal agar medium (CMA- biochemical and genetic characteristics not identical to known BBL, USA), containing 1% (v/v) of polysorbate 80 (tween 80) as yeast species also live. In fact, Cadez et al. (2012, 2013) described described by Kelly and Funigeillo (1959). The microscopic obser- new yeast species called Candida adriatica, Candida molendinolei, vations were carried out after 9 days of incubation at 25 C using an Ogataea kolombanensis, Ogataea histrianica and Ogataea deakii. Olympus mod. BX50F4 (Olympus Optical Co., Japan) light micro- These species have been isolated from olive oil and its by-product. scopy fitted with a camera. All the experiments were performed Ciafardini et al. (2013) report a new yeast species from Italian olive with three repetitions. The lipase tests were conducted both with oil, namely Yamadazyma terventina. Some of these new species synthetic and natural substrates. The trials with the synthetic such as C. adriatica and Y. terventina as well as C. diddensiae, were chromogen substrates were accomplished by transferring 30 mg of found frequently in extra-virgin olive oils of the Leccino variety each yeast culture biomass into the test tubes containing 10 mL of produced in central Italy, but the role that they play during the substrate. The synthetic chromogen substrates used were p-nitro- storage of the oil is not well-known. In fact, to date, it is not possible phenylbutirate, p-nitrophenylcaprate, p-nitrophenylcaproate and to know if their enzymatic activity is able to improve or not the p-nitrophenylpalmitate (SigmaeAldrich Co, USA). The trials were chemical and sensory characteristics of freshly produced olive oil carried out as described by Jin-Kyn et al. (2005) with some modi- during its storage. One of the possible negative effects of some fications. In detail, each substrate described above was dissolved in yeasts on olive oil quality refers to the increase in acidity promoted 400 mL of acetonitrile, then 40 mL of 2-N-morpholino ethane- by the lipase of microbial origin active on the triacylglycerol. sulfonic acid sodium salt (MES) hydrate (SigmaeAldrich Co, USA), However, at the present state of knowledge, it is still not completely 50 mM pH 6, was added to reach a final substrate concentration of clear whether the enzymatic degradation of the olive oil tri- 3 mM. Each solution was mixed briefly using a magnetic stirrer. It acylglycerols is due mainly to the lipases from the olive fruits was then transferred separately into the test tubes. Four repetitions crushed in the mill or by those of microbial origin produced by the and one negative control without the yeast biomass were accom- oil microbiota. Previous micro-biological research indicated that plished. The test tubes were swirled for 1 min with a vortex and þ certain Lip strains of Williopsis californica and Saccharomyces cer- then incubated in agitation (50 rpm) at 30 C. After 150 min of evisiae, inoculated in the same oil from which they were isolated, incubation the reaction was stopped without interfering with the increased the extra virgin olive oil acidity respectively from 0.62% reactants, by the addition of 50 mL of a 40% (w/v) NaOH solution. to 1.50% and 1.62% during a two-week incubation period. This The mixture was then centrifuged (Universal 32 centrifuge, Hettich, exceeded the limit of 0.8% established by current regulations for Tuttlinger, Germany) at 10,000 g for 5 min and the supernatants this commercial category of olive oil (Ciafardini et al., 2006a,b; EC, were analysed with a model 6300 Jenway spectrophotometer (U.K.) 2003, n. L. 295/57). Given that the lipase can have a negative effect at 410 nm, zeroing the instrument with the negative control on the quality of olive oil during its storage, a study has been without the yeasts' biomass. The lipase activity in the presence of þ conducted on the lipolytic activity of Lip strains of a yeast natural substrates was evaluated in sterile test tubes containing commonly found in Italian olive oil, namely C. diddensiae and other 10 mL of filter-sterilized extra-virgin olive oil using a white 47 mm two new yeast species recently discovered in olive oil, namely sterile filter with 0.45 mm of porosity (Millipore Co, MA 01821) and C. adriatica and Y. terventina. Both studies were carried out on olive 30 mg of the above yeast strains' biomass. The extra-virgin olive oil, oil characterized by a different polyphenols and water content. produced by the Leccino variety, was characterized by the following analytical indices: free fatty acids, 0.60%; peroxide values, 8.7 mEq 2. Material and methods 02 per kg oil; bitterness index 0.38; total polyphenols, 84 mg per kg oil and 0.31% of water content. Four repetitions and one negative 2.1. Enzymatic activity and yeast growth behaviour control without yeast were also carried out for each strain. The test tubes were swirled 3 min with a vortex and then incubated 40 days The yeast strains used in the trials had been previously isolated in a dark place at 30 C. At the end of the incubation the test tubes from extra-virgin olive oil and classed through physiological and were centrifuged at 10,000 g for 10 min and the oil was used for genetic analysis as C. adriatica (Cadez et al., 2012), C. diddensiae the chemical analysis of the acidity and fatty acid profiles. The (Zullo et al., 2010) and Yamadazima terventina (Ciafardini et al., acidity and the peroxide values were assessed according to the 2013) species. The yeasts were grown for 3 days at 30 C in Petri European Community regulation 299/2013 (EC, 2013). The profile dishes with MYGP agar medium containing: 3 g yeast extract of the free fatty acids of the same olive oil samples incubated for 40 (Biolife, Milan, Italy), 3 g malt extract (BBL, Cockeysville, MD, USA), days in the presence of the different yeast strains already described 5 g beef extract powder (BBL), 10 g D-glucose (Merck, Darmstadt, was made according to the European Community1429/92 Regula- Germany), 1000 mL distilled water, pH 7, as described by Kurtzman tion (EC, 1992). The analysis of fatty acid methyl esters was per- and Fell (1998). All the yeast cultures were further characterised by formed using a Fisons HRGC (Mega 2 series) with a flame-ionisation taking into consideration dimorphic growth behaviour and some detector and equipped with a WCOT fused-silica capillary column, important enzymatic activity involved in the hydrolysis of the FFAP-CB coating, film thickness 0.30 mm, 25 m in length 0.32 mm synthetic esters as well as the olive oil triacylglycerol compounds. i.d. from Chrompack (Middleburg, The Netherlands). The total The chromogenic characteristics of the yeast colonies were evalu- polyphenols were isolated three times by extraction of a solution of ated by cultivating the cells on the MYGP agar medium for 5 day at oil in hexane with a water-methanol mixture (40:60, v/v). The Download English Version: https://daneshyari.com/en/article/4362845

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